1. Field of the Invention
The present invention is directed to a method of synthesizing sulfurized oligonucleotide analogs by reacting an oligonucleotide analog containing a trivalent phosphorous linkage with a dithiocarbonic acid diester polysulfide.
2. Discussion of the Background
It is well-known that most of the bodily states in mammals, including most disease states, are effected by proteins. By acting directly or through their enzymatic functions, proteins contribute in major proportion to many diseases in animals and man.
Classical therapeutics has generally focused on interactions with such proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to directly inhibit the production of proteins involved in disease by interacting with the messenger RNA (mRNA) molecules that direct their synthesis. These interactions have involved the hybridization of complementary, or antisense, oligonucleotides or oligonucleotides analogs to mRNA. Hybridization is the sequence-specific hydrogen bonding of an oligonucleotide or oligonucleotide analog to an mRNA sequence via Watson-Crick hydrogen bond formation. Interfering with the production of proteins involved in disease would provide maximum therapeutic results with minimum side effects.
The pharmacological activity of antisense oligonucleotides and oligonucleotide analogs depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor for oligonucleotides and analogs thereof is their stability to nucleases. It is unlikely that unmodified oligonucleotides containing phosphodiester linkages will be useful therapeutic agents because they are rapidly degraded by nucleases. Modified oligonucleotides which are nuclease resistant are therefore greatly desired.
Phosphorothioate and phosphorodithioate oligonucleotide analogs which have one or both of the non-bridging oxygens of the natural phosphodiester linkage replaced with sulphur, respectively, are especially promising antisense therapeutics. These oligonucleotide analogs are highly resistant to nucleases, have the same charge as natural phosphodiester-containing oligonucleotides, and are taken up by cells in therapeutically effective amounts. See, for example, Baracchini et al, U.S. Pat. No. 5,510,239; Ecker, U.S. Pat. No. 5,512,438; Bennett et al, U.S. Pat. No. 5,514,788; and Ecker et al, U.S. Pat. No. 5,523,389.
Phosphorothioate and phosphorodithioate oligonucleotide analogs are conveniently synthesized with automated DNA synthesizers using hydrogen phosphonate chemistry which permits the phosphonate backbone to be sulfurized in a single step after automated synthesis. One drawback of this approach is that coupling yields during chain synthesis are typically lower than those obtained using phosphoramidite chemistry. The final yield of the desired oligonucleotide analog is therefore too low due to the low individual coupling yields.
Automated synthesis using phosphoramidite chemistry is a highly desirable approach to the synthesis of these sulfurized oligonucleotide analogs, with coupling yields typically greater than 99%. However, the phosphorous(III)-containing phosphite intermediates are unstable under the conditions of the detritylation step of the synthesis cycle. Therefore, these phosphorous(III) linkages must be sulfurized after each coupling step.
A more recent method for the synthesis of oligonucleotide analogs is the "blockmer" approach. In blockmer synthesis, an oligonucleotide analog is made by the sequential coupling of short protected oligomers or blocks, e.g., a dinucleotide, on a solid support. This strategy offers several advantages over the conventional synthetic approach which involves the sequential coupling of monomeric nucleoside phosphoramidites. The number of synthesis cycles required to prepare an oligonucleotide analog is reduced, saving time and minimizing reagent consumption. The blocks may be prepared on a large scale using inexpensive solution phase synthesis techniques. In order to prepare sulfurized oligonucleotide analogs by the blockmer method, a reagent for sulfuring the phosphorous(III) linkages of the blocks on a large scale is required. The blockmer approach is described in the following references: Ravikumar et al, WO 95/32980; WO 94/15947; Journal of Organic Chemistry 1984, 49, 4905-4912; Helevetica Chimica Acta 1985, 68, 1907-1913; Chem. Pharm. Bull. 1987, 35, 833-836.
There are several reagents available for sulfurizing the phosphite intermediates during automated oligonucleotide synthesis. All of these reagents have drawbacks which limit their use for synthesizing sulfurized oligonucleotide analogs.
Elemental sulfur, for example, has been used to sulfurize phosphorous(III) linkages in solid phase oligonucleotide synthesis. However, elemental sulphur is not suitable for use with automated synthesizers because of its poor solubility in standard solvents and slow sulfurization rate. In addition, carbon disulfide, the preferred solvent for elemental sulphur, is highly volatile and has a low flash point. See, U.S. Pat. No. 5,252,723 and U.S. Pat. No. 5,449,769.
The Beaucage reagent, 3H-1,2-benzodithiol-3-one, is a considerably more efficient sulfurizing agent. However, this reagent precipitates from solution and clogs the solvent and reagent transfer lines of an automated DNA synthesizer. Also, the by-product formed during the sulfurization reaction is a potent oxidizing agent. This by-product can lead to side products, e.g., phosphodiesters, which are difficult to separate from the desired sulfurized oligonucleotides. In addition, the preparation of this reagent involves expensive and toxic materials, and is therefore not amenable for large-scale synthesis of sulfurized oligonucleotide analogs. See, U.S. Pat. No. 5,003,097.
Tetraethylthiuram disulfide is an inexpensive and chemically stable sulfurization reagent. However, the sulfurization rate is slow and therefore a significant molar excess of this reagent is required. Even with an excess of this reagent, sulfurization yields are unacceptably low. See, U.S. Pat. No. 5,166,387.
Phenylacetyl disulfide may be used to sulfurize phosphite intermediates during automated oligonucleotide synthesis. However, this reagent has not been reported to be useful for large-scale synthesis of sulfurized oligonucleotide analogs. See, Recherches Travaux Chimiques des Pays-Bas 1991, 110, 325-331; Tetrahedron Letters 1989, 30, 6757-6760; Synthesis 1981, 637-638.
Accordingly, there remains a need in the art for methods and reagents for synthesizing sulfurized oligonucleotide analogs which overcome these problems.